Dyes have applications in many technologies, both new and well established, which count amongst them textile dyeing, optical data storage media and various detection methods, for both chemical and biological use. Within these applications various properties of the dyes are exploited in their actual detection which may ultimately be traced back to absorption, reflectance and luminescence (chemiluminescence, fluorescence and phosphorescence). However, a recurring theme of many applications is the need to form a relatively permanent link, in the form of a covalent bond, between the dye and the substrate of choice. The substrate may be inter alia: a relatively inert surface or probe possibly of insoluble polymeric origin; a macromolecule of biological origin e.g. a protein, antibody or oligonucleotide; or a small molecule of either biological or synthetic origin e.g. a hapten for use in directed or molecular recognition based applications; or a molecule which may modify the properties of the dye e.g. a water solubilising group such as a carbohydrate or other polar residues; or a species which protects the dyes from or decreases the rate of photofading of the dye, such as hindered amines and various transition metal complexes; or possibly another dye conjugate system thus enabling the possibility of energy transfer or fluorescence quenching based applications. The requirement for a covalent linkage means that many commercial products, spanning a large range of dye classes and possessing the desired spectral properties, cannot be used. This therefore necessitates either the modification of an existing product or the complete synthesis from appropriate materials such that a suitable grouping is available for linking the dye and substrate. There are many different sorts of linkage known, the main requirements being that the linkage is relatively stable and that it is easily introduced in high yield or efficiency. One of the most versatile groupings is that of the carboxylic acid which is readily converted to a range of reactive species or used to form a link to another substance containing the reactive linking species of choice.
This invention is concerned with a relatively simple and easily prepared dye intermediate and its use for preparing a range of dyes particularly fluorescent dyes having reactive groups by which they can be linked to various substrates. The intermediate is a m-aminophenol having the formula (I) 
Where
R1 is xe2x80x94RaRb, where Ra is C1-C10 alkylene or aralkylene which may optionally contain 1 to 4 oxygen, nitrogen or sulphur atoms in a straight or branched chain, and Rb is carboxy including acid, is salt, ester including N-hydroxysuccinimide, activated ester or amide,
R2, R3, R4 and R5 are the same or different and each is: H; or C1-C10 alkyl or aralkyl which may optionally contain 1 to 4 oxygen, nitrogen or sulphur atoms in a straight or branched chain; or Rc where Rc is a group to modify solubility, or a group to modify electronic and/or spectral properties, or a functional linking group; or xe2x80x94RaRc wherein Ra and Rc are herein defined,
or R4-R5 and/or R2-R4 and/or R2-R3 are linked to form an extended ring system, carbocyclic or heterocyclic, aromatic or aliphatic which is unsubstituted or substituted as above,
R6 is H or xe2x80x94CR9O or Nxe2x80x94O.
R9 is H, C1-C6 alkyl phenyl or RaRc wherein Ra and Rc are herein defined.
R1 may be different or the same as R2.
R2, R3, R4 and R5 may contain carbon chains which are linked to form extended ring systems, either aliphatic or aromatic, for example 
where
each of R7 and R8 are defined as R2, R3, R4 and R5.
Many of the dyes described below are made from compounds of formula (I) in which R6 has been changed from H to either formyl or nitroso, for example 
These may be made from the corresponding compounds where R6 is H by standard methods.
Groups to modify solubility include phosphate, sulphonate, carbohydrate, poly(oxyethylene) and perfluoroalkyl. Groups to modify electronic and/or spectral properties include nitro, cyano, halogen and alkoxy. Functional groups for linking to another component include carboxylic acid or derivative or activated ester, azide, amine, hydroxyl, sulphonyl chloride, isothiocyanate, phosphoramidite, vinyl sulphone, maleimide, halotriazine, iodoacetamide and N-hydroxysuccinimide; see also Andrew Garman: xe2x80x9cNon-Radioactive Labelling: A Practical Introductionxe2x80x9d published by Academic Press, London, 1997, and references cited therein.
These compounds may be linked, preferably through Rb or alternatively through R2, R3, R4, R5, R7 or R8 to a support such as polyethylene glycol, polyethyleneimine, polysaccharides such as dextran, or a derivatised substrate such as that used in solid phase synthesis e.g. polystyrene, polyacrylate or glass.
Preferably Ra is C1-C4 alkylene. Certain of these compounds can be made by an advantageous process which forms another aspect of this invention, and which comprises reacting 
with GG1 
where
G is CHR=CRxe2x80x94 where R is H or C1-C4 alkyl
and G1 is an electron withdrawing group for example CN or carboxy where the carboxy group is acid, salt, ester or amide.
Other compounds may be made by the use of G2RaG1 where G2 is a leaving group and Ra and G1 are herein defined.
An intermediate compound of formula (I) may be immobilised on a support and there reacted to form the desired dye. This method may give access to a combinatorial library of dyes or an easy way of purifying dyes, or may permit the labelling of a first nucleotide or amino acid of a solid phase oligonucleotide or oligopeptide synthesiser.
In one aspect, this invention provides use of the said intermediate to make a dye selected from a defined group, or a leuco-dye or reduced analogue of said dye. In another aspect, the invention provides the resulting dyes, and their leucodye analogues, as new compounds. The dyes are as follows (in each case, R1, R2, R3, R4 and R5 are as defined above for the intermediate compound (I); and may be the same or different at different parts of the molecule): 
where
X is O, NH or =NCOAr
X1 is H, CN, CHO, CH=N+(RdRe), NO, COOH, COORf, CONRgRh, C1-C10 alkyl, aralkyl or aryl or 
where
X3 is NH, N-Rk, O or S, wherein X1 is either unsubstituted or substituted by: a group to modify solubility; or a group to modify electronic and/or spectral properties; or a functional group for linking to another component,
Rd and Re are alkyl, aryl, aralkyl,
Rf, Rg, Rh and Rk are RaRc wherein Ra and Rc are herein defined,
X2 is H, CN, ORi, Cl, Br, alkyl or aryl which maybe unsubstituted or substituted by: a group to modify solubility; or a group to modify electronic and/or spectral properties; or a functional group for linking to another component;
where Ri is MeC6H4SO2xe2x80x94, CH3SO2xe2x80x94, P=O(ORj)2 
Rj is alkyl, aryl, aralkyl
X1 and X2 may contain atoms which are linked to form a carbocyclic, heterocyclic, aliphatic or aromatic ring system which may be substituted or unsubstituted with a group to modify electronic and/or spectral properties and/or a group to modify solubility properties and/or a functional linking group;
provided that, when X is O, then X1 is not H, CN, or C1-C10 alkyl, aralkyl or aryl; and provided that when X is O, then X3 is not S; 
where
Y is CN or CONH2 or CH2NH2,
Y1 is NH or O or =NCOAr,
Y2 and Y4 are: H; or C1-C10 alkyl or aralkyl which may optionally contain 1 to 4 oxygen, nitrogen or sulphur atom in a straight or branched chain; or Rc where Rc is a group to modify solubility, or a group to modify electronic and/or spectral properties, or a functional linking group; or xe2x80x94RaRc wherein Ra and Rc are herein defined,
Y3 is H or CN; 
where
R10, R11, R12, R13 and R14 are the same or different and each is: H; or C1-C10 alkyl or aralkyl which may optionally contain 1 to 4 oxygen, nitrogen or sulphur atoms in a straight or branched chain; or Rc where Rc is a group to modify solubility, or a group to modify electronic and/or spectral properties, or a functional linking group; or xe2x80x94RaRc wherein Ra and Rc are herein defined,
R10 and R11; and/or R13 and R11; and/or R12 and R14 may be linked to form an extended ring system, carbocyclic or heterocyclic, aromatic or aliphatic which is unsubstituted or substituted as above,
Z is aryl which is unsubstituted or substituted by: a group to modify solubility; or a group to modify electronic and/or spectral properties; or a functional group for linking to another component, provided that Z is not 2-carboxy-3,4,5,6-tetrachloro phenyl; 
xe2x80x83provided that NR13R14 is different from NR1R2; 
where
A is N or CH
A1, A2A3 and A4 are the same or different and each is: H; or C1-C10 alkyl or aralkyl which may optionally contain 1 to 4 oxygen, nitrogen or sulphur atom in a straight or branched chain; or Rc where Rc is a group to modify solubility, or a group to modify electronic and/or spectral properties, or a functional linking group; or xe2x80x94RaRc where Ra and Rc are herein defined; 
where
n is 0, 1 or 2, B1 is H, C1-C10 alkyl, aryl or aralkyl either unsubstituted or substituted by a group to modify solubility or a group to modify electronic and/or spectral properties, or a functional linking group, Cl, Br or F,
B2 and B3 are the same or different and are selected from C1-C10 alkyl, aryl or aralkyl either unsubstituted or substituted by a group to modify solubility or a group to modify electronic and/or spectral properties, or a functional linking group,
B1 and B2 and/or B1 and B3 and/or B2 and/or B3 may be linked to form an extended ring system, carbocyclic or heterocyclic, aromatic or aliphatic which is unsubstituted or substituted by a group to modify solubility, or a group to modify electronic and/or spectral properties, or a functional linking group; 
where
D, D1, D2 and D3 are the same or different and each is: H; or C1-C10 alkyl or aralkyl which may optionally contain 1 to 4 oxygen, nitrogen or sulphur atom in a straight or branched chain; or Rc where Rc is a group to modify solubility, or a group to modify electronic and/or spectral properties, or a functional linking group; or xe2x80x94RaRc where Ra and Rc are herein defined; 
where
E1 is H, C1-C10 alkyl, aryl or aralkyl either unsubstituted or substituted by a group to modify solubility or a group to modify electronic and/or spectral properties, or a functional linking group, Cl, Br or F,
E2 and E3 are the same or different and are selected from C1-C10 alkyl, aryl or aralkyl either unsubstituted or substituted by a group to modify solubility or a group to modify electronic and/or spectral properties, or a functional linking group,
E1 and E2 and/or E1 and E3 and/or E2 and/or E3 may be linked to form an extended ring system, carbocyclic or heterocyclic, aromatic or aliphatic which is unsubstituted or substituted by a group to modify solubility, or a group to modify electronic and/or spectral properties, or a functional linking group; 
where
R15, R16, R17, R18, R19, R20, R21, R22, R23 and R24 are the same or different and each is: H; or C1-C10 alkyl or aralkyl which may optionally contain 1 to 4 oxygen, nitrogen or sulphur atoms in a straight or branched chain; or Rc where Rc is a group to modify solubility, or a group to modify electronic and/or spectral properties, or a functional linking group; or xe2x80x94RaRc wherein Ra and Rc are herein defined,
R16 and R17; and/or R21 and R22; and/or R15 and R18 and/or R16 and R19; and/or R20 and R23; and/or R21 and R24 may be linked to form an extended ring system, carbocyclic or heterocyclic, aromatic or aliphatic which is unsubstituted or substituted as above.
By virtue of the reactive group R1, the dyes of the present invention may be combined with target materials to form conjugates. Suitable target materials may include antibodies, antigens, proteins, carbohydrates, polysaccharides, lipids, nucleotides, nucleic acids, polymer particles or surfaces and glass beads and surfaces. Thus, for example, the carboxyl group of R1 may be converted to an N-hydroxysuccinimidyl ester for linking with an amino group of a polypeptide. These methods and the products resulting from them, either dye-labelled biomolecules or dyes immobilised on particulate or massive surfaces, are envisaged as further aspects of the invention.
The main uses for the dyes of this invention are expected to be for labelling biologically active molecules such as nucleotides, oligo- and poly-nucleotides, nucleic acids and nucleic acid analogues (e.g. PNA), amino acids, peptides, proteins, antibodies, haptens, etc. for use in biological detection systems. Alternatively, the dyes may be conjugated to species which can direct the path of the dye within or aid entry to or exit from cells (live or dead); such as for example long alkyl residues to allow permeation of lipophilic membranes, or intercalating species to localise a dye in a nucleus or other cellular enclave containing double-stranded DNA or in vitro applications involving double-stranded nucleic acids. Dyes may also be immobilised on substrates such as particles or beads. The dyes may be involved in a resonance energy transfer system of the kind described in EPA 747 700.
The actual properties of the dyes used as detection labels vary. The observed property may simply be colour (based on light absorbance), luminescence or fluorescence (based on light emission) or other properties such as fluorescence lifetimes or degree of polarisation of emitted light. The dyes may also be used outside biological detection systems. For example, a dye may be immobilised on a probe for use in a chemical detection system e.g. a colour change, pH probe, or a chemical detector based on solvatochromism, electrochromism, thermochromism or photochromism. Reactive dyes may also be useful in printing e.g. in order to obtain improved substantivity effects.
There follow a number of example applications, which indicate a general chemical strategy by which intermediate compounds of formula (I) are used to make various families of dyes. The lettering system used is consistent within any particular application, but may not be consistent with that used in the introduction above and the claims below.
Application 1: Green-fluorescent Coumarin Dyes 
If X is simply xe2x80x94CN or an aryl substituent then the dyes are colourless with a blue fluorescence emission. To obtain yellow dyes with a green fluorescence the moiety X is a benzimidazole, benzoxazole or benzthiazole ring system, with or without additional substituents (e.g. halogen, sulphonic acid).
Application 2: Yellow and Orange Fluorescent Coumarin Dyes 
Application 3: Red-fluorescent Benzopyrano-benzimidazo-pyridines 
Substituents R in the benzimidazole ring may be H or any common substituent, but most usefully could be xe2x80x94SO3H to enhance water solubility or a grouping capable of forming a linkage, e.g. xe2x80x94CO2H, to give a bis-functional dye.
Application 4: Red-fluorescent Rhodamine Dyes 
As shown above, such dyes may be symmetrical or unsymmetrical. There is the problem of the additional carboxylic acid group in the meso phenyl ring, which should preferentially be esterified. Alternatively, sulphorhodamines or non-meso-carboxylated analogues could be considered: 
Application 5: Near-infrared Absorptive Croconium Dyes 
Use of this type of intermediate (i.e. with a meta-hydroxy group) is essential for the success of the reaction with croconic acid. If the dyes are fluorescent, then the emission will probably lie beyond the range of most fluorimeters, probably near 900 nm.
Application 6: pH Sensitive Photochromic Dyes 
Application 7: pH Sensitive Green-black Fluorans 
The open chain form of the dye is not fluorescent and is usually very dark in colour due to its multiple absorption band chromophore.
Application 8: Styryl Hemi-cyanines 
Application 9: Leuco Dyes for Use as Enzyme Substrates
These are not dyes, as such, but a reduced, or leuco, form of those already listed which for example, may be chemically or enzymatically oxidised back to parent dye molecule. The dyes may be derivatised slightly to increase their stability.
Application 10: Oxindigo Derivatives 
These dyes can have large Stoke""s shift (50-100 nm) emitting in the red, xcexmax(Abs.) 510-530 nm, xcexmax (Em.) 600-620 nm. They are also reported as being strongly fluorescent with high colour fastness. (Angew. Chem. Int. Ed. Engl. 1996, 35 (9) 1016), and therefore the ability to sum multiple scans.
Application 11: Squarylium Dyes 
Application 12: 5,10,15,20-Tetraarylporphyrin Dyes 
with or without a central metal ion.